Infections by Gram-positive bacteria cause tens of thousands of deaths in the United States each year. The transcriptional regulator catabolite control protein A (CcpA) has recently been found to influence the virulence gene expression profile during growth in standard laboratory medium of numerous Gram-positive pathogens including group A Streptococcus (GAS). CcpA was originally identified in Bacillus species where it binds DNA sequences termed catabolite response element (cre) sites in conjunction with its co-factor histidine-containing phosphoprotein phosphorylated at serine residue 46 (HPr-Ser46-P). Orthologues of CcpA, HPr, and the kinase/phosphorylase that regulates HPr phosphorlyation status (HPrK/P) are present in all major Gram- positive human pathogens, and there are accumulating data suggesting that the CcpA-(HPr-Ser46-P)-HPrK/P axis contributes to the infectivity of diverse bacterial species. The long term goals of this project are to clearly define how the CcpA-(HPr-Ser46-P)-HPrK/P axis contributes to GAS virulence and to ascertain mechanisms by which CcpA influences gene expression during infection. The specific aims of this proposal have been designed to test hypotheses arising from preliminary data demonstrating that: 1) Inactivation of CcpA altered GAS gene expression during infection in mouse muscle; 2) Inactivation of CcpA decreased GAS virulence in a mouse myositis model; 3) Mutation of the central CpG of a GAS cre site decreased CcpA-(HPr-Ser46-P) DNA binding affinity; and 4) CcpA influenced gene expression under conditions in which HPr-Ser46-P levels are low or absent. In aim 1, gene expression analysis of wild-type and CcpA-inactivated GAS strains will be performed in multiple mouse models of infection to test the hypothesis that CcpA is a key virulence factor regulator during GAS infection. In aim 2, the virulence of CcpA isogenic mutant strains derived from different M protein serotype parental strains will be compared to their parental wild-type strains in various mouse models of infection to test the hypothesis that CcpA positively contributes to the infectivity of GAS strains of multiple M protein serotypes. In aim 3, a combination of in vitro and in vivo methods will be used to test the hypothesis that the central cre site CpG is critical for the effect of CcpA on GAS gene expression. In aim 4, the gene expression profiles of CcpA and HPr-Ser46-P inactivated strains will be compared under different metabolic conditions to test the hypothesis that CcpA can affect GAS gene expression in the absence of HPr-Ser46-P. The highly conserved nature of the CcpA-(HPr-Ser46-P)-HPrK/P axis amongst major Gram-positive bacterial pathogens means that the successful completion of the proposed research may generate new insights into disease pathogenesis that are relevant to a broad array of pathogenic bacteria. Such information could form the basis for the development of novel therapeutic or preventive interventions.